Optimization And Mechanism Of Immune Strategy For Gene Cancer Vaccine

Cancer is expected to rank as the leading cause of death among all the noncommunicable diseases?NCDs?.Due to the devlopment of medical technology and and awareness of health life,early diagnosis and treatment of cancer cases increased,the mortality of cancer decreased.However,here is still a high mortality rate of recurrence and metastasis of cancer patients.Gene cancer vaccine is a kind of tumor immunotherapy.Tumor-associated antigens?TAAs?or tumor-specific antigens?TSAs?are constructed into vectors of DNA plasmid,RNA or virus.DNA vaccine and recombinant adenovirus vaccine are widely used for their safety.However,due to its weak immunogenicity,DNA vaccines need to be enhanced by adjuvants,electroporation and other methods.The gene vaccines used in this research consist of a DNA vaccine and a recombinant adenovirus vaccine.The DNA vaccine?CpDV-IL2-sPD1/MS?was constructed with two TAAs survivin and MUC1 gene sequences in the double-cistron–expression-vector,to enhance DC targeting,soluble PD1 sequence was added.IL-2 and CpG motif were constructed as immune adjuvants.The recombinant adenovirus vaccine AD-MS was constructed based adenovirus type 5vector and inserted survivin and MUC1 fusion gene.In our previouse studies,DNA vaccine prime/recombinant adenovirus vaccine boost strategy has achieved good anti-tumor effects on murine lung cancer model.In this reseach,the immunization strategy was further optimized to enhance the anti-tumor effect.To accelerate immune response,a rapid immunization strategy was adopted.The interval of DNA vaccine immunization was shortened from 1-2 weeks to 3 days.AD-MS was vaccinated to boost the immune response.The results showed that compared with the conventional immunization strategy,DNA vaccine under the fast immunization stretegy induced higher CTL rate,and a 4 to 5 fold of IFN-?secretion.In the lung cancer mice model,DNA vaccine under normal immunization was vaccinated 1days after tumor challenge.Under the fast immunization strategy,DNA vaccine was given 8 days after the tumor challenge.The inhibition rate of normal immunization group was 52.68%,and that of the rapid immunization group was47.82%.The tumor growth was significantly inhibited in both groups?P<0.01?,but there was no significant difference between the two groups.The life prolongation rate of the rapid immunization group was 47.75%,while that of the nornal immunization group was 27.96%,life time of tumor-bearing mice in rapid immunization group was prolonged significantly?P<0.05?.It can be seen that the rapid immunization group induced a stronger immune response,even for the late course of tumor,it still had a better anti-tumor effect.To investigate the mechanism of rapid immunization strategy,the expression of CCL3 and CCL5 in the muscle tissue at the injection site was detedted,the data showed that the expression of CCL3 and CCL5 increased slightly 24-48 hours after immunization,and the accumulation of CD8⁺,CCR5⁺and CCR6⁺DC cells at the injection site was detected 2 days after immunization.The expression peak of CCL19 and CCL21 in the lymph nodes appeared 72 hours after immunization.The2^nd vaccination was gived 48h after the first vaccination when immature DCs stayed at the injection site.It could increase the uptake of DNA plasmids or antigen expressed by DNA vaccine,which will help induce a higher cellular immune response.Based on this principle,the rapid immunization strategy was further optimized for DNA immunization on days 0,2 and 5,and the immunization began after tumor formation.Within 3-14 days after immunization,the DNA vaccine with rapid immunization strategy had showed a better antitumor effect.The tumor inhibition rate reached 83.6%?P<0.01?on the 3^rd day and 46.34%?P>0.05?on the14^th day after immunization.The immune response data showed that the immune peak appeared 7-10 days after immunization,and the proportion of CD8⁺T lymphocytes in spleen and in tumor tissue gradually decreased after the immune peak.The use of electroporation further enhanced the immune response of vaccines.The results showed that the electroporation could accelerate the expression of the DNA vaccine,but not the total protein expression.In addition,electroporation could promote the accumulation of CCR5⁺and CCR6⁺DC cells at the injection site to improve the vaccine effect.In addition,the same level of immune response could be induced only by electroporation duiring the first DNA immunization under the rapid immunization strategy.To eliminate immunosuppressive factors,the vaccine was combined with gemcitabine/cisplatin which is applied in lung cancer treatment.The results showed that,in high dose gemcitabine?60mg/kg?/cisplatin?4mg/kg?goup,the ratio of MDSC was reduced by 54.44%compared with the control group.Although the high dose gemcitabine/cisplatin killed CD8⁺T lymphocytes,it could significantly promote the infiltration of CD8⁺T lymphocytes into tumor tissues?7.93%,about4.08 times of the control group?;low dose gemcitabine?10mg/kg?/cisplatinum?0.667 mg/kg?significantly reduced the proportion of MDSC in spleen cells?40.24%lower than that in control group?,but did not kill CD8⁺T lymphocytes in spleen cells,but the antitumor effect was significantly lower than that in high dose group?P<0.05?.Therefore,two doses of chemotherapeutics were combined with different immunization strategies to evaluate their antitumor effects.High dosage of gemcitabine/cisplatin had significant effect on effector cells,CD8⁺T cells.Therefore,the vaccine was vaccinated after two cycles of chemotherapy.The results showed that the life-prolonging rate of the tumor-bearing mice was 131.48%when the vaccine combined with high dosage of chemical drugs.CD8⁺T lymphocytes in the combined group were 12.5 times higher than those in the vaccine group;CD8⁺T lymphocytes in the spleen cells were increased by 31.78%;Treg and MDSC in the spleen cells were significantly reduced by 40%and 50%when compared with the vaccine group.In low dose gemcitabine/cisplatin and vaccine combined group,DNA vaccine was vaccinated under normal immune strategy,low dose gemcitabine/cisplatin was treated during the vaccination.The tumor inhibition rate of the combined group was69.31%,twice as high as that of the vaccine alone,and the life prolongation rate was increased from 31.4%to 55.56%.The infiltration of CD8⁺T lymphocytes in tumor tissue was 5.18 times higher than that in vaccine alone group.The proportion of Treg in splenocytes of the combined group decreased by 58.18%compared with that in the vaccine group.In summary,the optimized rapid immunization strategy,three times of DNA vaccine are given 2 days and 3 days intervals,adenovirus vaccine is given 10 days after thr 3^rd DNA vaccination,can induce immune response effectively.Vaccines given under fast immune strategy could still suppress the growth of tumor,which is in a late couse of disease.Electroporation can enhance the vaccine effect and induce higher immune response.Rapid immunization strategy and electroporation can enhance the uptake of DNA vaccine or expressed antigen by DC by promoting the migration of CD8⁺,CCR5⁺,CCR6⁺DC cells to the injection site,thereby enhancing the immune response of the vaccine.High dose of gemcitabine?60mg/kg?/cisplatin?4mg/kg?and low dose of gemcitabine?10mg/kg?/cisplatin?0.667mg/kg?promote the infiltration of CD8+T cells in tumor tissue and enhance the anti-tumor effect by reducing the proportion of Treg and MDSC.The antitumor effects of the two combined strategies were significantly higher than those of the respective vaccines alone.